The goal of this proposal is to investigate the mechanism of cell cycle regulation during the mammary gland development and the consequence of its alteration during breast tumorigenesis. In particular, we will initiate studies using mouse as an animal model system to determine the roles of recently identified SCF SKP2 complex (Skp1, Cullins/CDC53, F-box proteins), an ubiquitin E3 ligase, during the G1/S transition and the tumorigenic potential of the pathogenic alteration of SCFSKP2 expression. We have originally isolated p45SKP2 as a protein that is highly induced in many transformed tumor cells. Our studies suggest that SKP2 is a component of SCFSKP2 ubiquitin E3 ligase and is required for the S-phase entry. We found that SKP2 specifically binds to and targets the CDK inhibitor p27Kip1 for ubiquitin- dependent proteolysis during the G1/S transition. Expression of SKP2 is sufficient to promote p27 degradation, CDK activation and S-phase entry in quiescent cells. p27 is a major cell cycle regulator that is controlled by a variety of signaling processes including cell attachment and soluble growth factors. p27 is primarily controlled at the level of protein stability and its degradation is required for the G1/S transition. Several tumor suppressors, including PTEN, have been implicated in p27 regulation. The down-regulation of p27 is correlated with malignancy of a wide variety of human cancers, including breast cancers. Studies suggest that loss of p27 is associated with poor survival of many young breast cancer patients. To investigate the regulation of breast tissue proliferation, differentiation, and the mechanisms that cause breast cancer, we propose to determine the role of SKP2 in mammary gland development and to examine its tumorigenic potential using the mouse transgene and knockout techniques. Our specific aims are: 1) to analyze the expression of SKP2, p27, cyclin D and other cell cycle regulators during mammary gland development. 2) To construct a transgenic mouse line to specifically express SKP2 in breast tissues to determine its effect on proliferation, differentiation, and potential tumorigenicity of the mammary gland. 3) To make a conditional SKP2 knockout construct in mice and to examine knockout phenotypes on breast tissue and mammary gland development. These studies should help to establish mouse models for breast cancer research and to reveal novel insights into the mechanism by which proliferation and tumorigenic processes of mammary gland are controlled during normal mammary gland development and the effect of its pathogenic alteration. These studies should also provide a basis to design novel strategies for diagnosis, prognosis, and therapeutic treatment of breast cancers.